Immunoblotting was performed as previously described [21 (link)]. Cells were prepared by RIPA (radio-immunoprecipitation assay) buffer with phosphatase inhibitors (Sigma). After vortex 30 s, cells were lysed at ice for 15 min and centrifuged at 12000 rpm for 15 min at 4°C. Supernatant was denatured and used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad; Hercules, CA, USA). Subsequently, the membranes were immunoblotted with the corresponding primary antibodies followed by peroxidase-conjugated secondary antibodies. The bonds were visualized by Chemiluminescence (Thermo Scientific; Waltham, MA, USA). SPOP (1:1000 Proteintech; 16750-1-AP), Flag (1:1000 Proteintech; 66008-2-Ig), GAPDH (1:2000 Cell Signaling Technology; #5174), FH (1:1000 Proteintech; 10966-1-AP), ELOVL2 (1:500 Abcam; EPR11880), ACADL (1:1000 Proteintech; 17526-1-AP) and Vinculin (1:2000 Sigma; V4505) antibodies were used in the study.
V4505
Manufactured by Merck Group
Sourced in Canada
The V4505 is a versatile laboratory equipment product manufactured by Merck Group. It is designed to perform essential tasks in various research and analytical settings. The core function of the V4505 is to provide a reliable and efficient platform for conducting experiments and analyses. The detailed specifications and intended applications of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (2 mentions)
Sc 271800, by Santa Cruz Biotechnology (2 mentions)
Mab5457, by R&D Systems (2 mentions)
Sc 517107, by Santa Cruz Biotechnology (2 mentions)
Sc 377120, by Santa Cruz Biotechnology (2 mentions)
Fluostar omega microplate reader, by BMG Labtech (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phospho ampkα, by Cell Signaling Technology (1 mentions)
Sc 25778, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab124759, by Abcam (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
F3648, by Merck Group (1 mentions)
Ab134047, by Abcam (1 mentions)
19 protocols using v4505
1
Western Blot Analysis of Cellular Proteins
2
Protein Extraction and Western Blot Analysis
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Total proteins from different organs or from adipocytes were extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, pH 8.0, 50 mM NaF, 1 mM sodium glycerophosphate, 5 mM pyrophosphate, 0.27 M sucrose, 1% Triton X-100, 0.1 mM PMSF, 0.1% 2-mercaptoethanol, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and centrifuged at 8,600 × g for 20 min at 4°C. Extracts were separated by SDS-PAGE and transferred to 0.2-µm-pore-size nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibodies to phospho-JNK (Thr183/Tyr185; 4668), total JNK (9252), phospho-AMPKα (Thr172; 2531), total AMPKα (2603), and phospho-p38 (Thr180/Tyr182; 9211; all from Cell Signaling Technology), p38 from Santa Cruz Biotechnology (sc-535), and GAPDH (sc-25778) and vinculin (V4505; Sigma-Aldrich). All antibodies were used at 1:1,000. After washes, membranes were incubated with an appropriate horseradish peroxidase–conjugated secondary antibody (GE Healthcare), and signals were detected using an enhanced chemiluminescent substrate (Clarity Western ECL substrate; Bio-Rad). Protein levels were analyzed by optical density measured with ImageJ (National Institutes of Health). Results were normalized to the not phosphorylated form.
3
Signaling Pathway Protein Analysis
4
Dystrophin Quantification in Muscle Tissues
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Protein extracts from tissues and cultured cells were prepared using a RIPA buffer (Cell Signaling). Protein concentration was determined using the BCA assay (Pierce). Fifty micrograms of total protein per lane were used for differentiated MPCs and muscles from injected adult mice. Samples were denatured at 99°C for 5 minutes before being loaded on to 3%–8% TA precast gels (Invitrogen). Dystrophin and vinculin (loading control) were detected by primary antibodies Mandra1 (1:100, Abcam) and V4505 (1:1,000, Sigma), respectively, followed by horse anti‐mouse IgG HRP‐conjugated secondary antibody (1:1,000, Cell Signaling Technology). A ChemiDoc imaging system (Bio‐Rad) was used to detect chemiluminescence after using a Supersignal West Dura ECL kit (Thermo Fisher). Intensities of dystrophin and vinculin bands were quantified using ImageJ (NIH) and the gel analysis function.
5
Protein Expression Analysis by Western Blot
6
Immunoblotting Analysis of Protein Lysates
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Total lysates were obtained homogenizing tissue samples in a potter with lysis buffer 1% Triton X-100, 0.5% sodium deoxycolate, 0.2% SDS, 150 mM NaCl, 2 mM EDTA, 10 mM HEPES (pH 7.4), 20 mM NaF, 1 mM orthovanadate and a protease inhibitor cocktail, for 30 min at 4 oC. After centrifugation at 10 000 g for 10 min at 4 °C, the supernatant was removed and stored at −80 °C. For immunoblotting equal amounts of protein (30 μg/lane) from tissue lysates were separated on 7.5% SDS–PAGE and transferred onto nitrocellulose sheets. The membranes were blocked and incubated overnight with primary antibodies GFAT1 and GFAT2 (SC134894 and SC134710, respectively, Santa Cruz Biotechnology), CTD-110.6 for O-GlcNAc (SC-59623, Santa Cruz Biotechnology), β-actin (A5316, Sigma Chemical) and vinculin (V4505, Sigma Chemical). After washing, the membranes were incubated for 1 h with peroxidase-conjugated secondary antibodies, developed using ECL (GE Healthcare, Pittsburgh, PA, USA) and exposed to Image Quant LAS 4000 (GE Healthcare). ImageJ software was used for densitometry analysis of immunoblots, and measurements were normalized against β-actin or vinculin loading controls.
7
Immunofluorescence Staining of Vinculin
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Cells were seeded on coverslips in a 12-well plate and incubated overnight in RPMI 1640 supplemented with 10% FBS and 1% P/S. Cells plated on coverslips were incubated with 4% paraformaldehyde for 10 mins at room temperature and washed three times with 1× PBS. Cells were then incubated with 0.5% Triton X-100 for 2 mins at room temperature and washed three times with 1× PBS. Coverslips were incubated with blocking buffer for 30 mins at room temperature. Coverslips were then incubated with mouse anti-vinculin antibody (Sigma-Aldrich, V4505, batch #0000216740, 1:500) overnight at 4°C. Coverslips were then washed three times with 1× PBS and incubated with Alexa Fluor 488-conjugated anti-mouse antibody (Thermo Fisher Scientific, A21202, 1:500) and Alexa Fluor 647-conjugated Phalloidin (Thermo Fisher Scientific, A22287, 1:500) for 30 mins at room temperature. After three washes in 1× PBS, cells were incubated with DAPI (Cell Signaling Technology, 4083S, 300 nM) for 5 mins. Coverslips were then washed twice with 1× PBS and once with distilled water before mounting the coverslips onto microscopy slides using 4 µl Mowiol. Coverslips were imaged on a Zeiss Axioplan2 microscope with a 63×/1.4 NA Plan-Achromat objective and a Photometrics Cool Snap HQ cooled charge-coupled device camera. Data for 20-30 cells per sample were analyzed using FIJI.
8
Comprehensive Antibody Validation for WB and IF
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Antibodies used for WB and immunofluorescence: anti-YAP is sc101199 (1:1,000, Santa Cruz Biotechnology), anti- GR is BK3660S (1:1,000, Cell Signaling), anti-actin is C11 (1:2,000, Sigma), anti-ANKRD1 is 11427-1-AP (1:1,000, Proteintech (DBA), anti-pYAP (Ser127) is 4911S (1:1,000, Cell Signaling), anti-WWTR1 is HPA007415 (1:1000, Sigma), phalloidin is A12379 (1:250, Alexa Fluor), anti-PARP-85 is TB273 (1:500, Promega), anti-vinculin is V4505 (1:4,000, Sigma), anti-LATS1 is ab70562 (1:500, Abcam), phospho-LATS1 (Thr1079) is BK8654S (1:500, Cell Signaling), anti-FAK (C-20) is sc-558 (1:1,000, Santa Cruz), anti-FAK (phospho Y397) is ab81298 (1:1,000, Abcam), anti-Src is BK2110S and anti-Phospho-Src (Tyr416) is BK2101S from Cell Signaling (1:1,000), anti-FN1 is GTX112794 (1:1,000, GeneTex), anti-Slug is C19G7 (1:500, Cell Signaling), anti-Mst1 is 3682S (1:500, Cell Signaling) and anti-phospho-Mst1/2 (T183/T180) is 3681S (1:500, Cell Signaling).
9
Analyzing HGF Adhesion on Topographies
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HGFs were seeded on each topography at 10,000 cells for 24 h and 1 week. At the end of the designated timepoints, samples were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 1% bovine serum albumin. Fixed and permeabilized cells were labeled with antibodies against fibronectin (sc-8422; Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000), α-SMA (A5228; Sigma-Aldrich, Oakville, ON, Canada; 1:1000), vinculin (V4505; Sigma-Aldrich; 1:100, Oakville, ON, Canada), and phospho-SMAD3 (phospho Ser423/Ser425) (ab52903; Abcam 1:200). These signals were detected using primary antibodies followed by species appropriate IgG conjugated to appropriate Cy5, FITC or TRITC secondary antibodies (Molecular Probes; 1:200 dilution). Images were taken on a Carl Zeiss Observer Z1 microscope using AxioVision Relative software (version 4.8, Toronto, ON, Canada).
10
Comprehensive Mitochondrial Protein Analysis
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For Western blot analysis, the following antibodies were used in a 1:1000 dilution: anti-β (ab14730, Abcam), anti-γ (PA5-29975, ThermoFisher), anti-b (ab117991, Abcam), anti-OSCP (ab110276, Abcam), anti-f (ab200715, Abcam), anti-g (ab126181, Abcam), anti-e (ab122241, Abcam), anti-c (ab181243, Abcam), anti-a (ab192423, Abcam), anti-citrate synthase (ab96600, Abcam), anti-vinculin (V4505, Sigma), anti-prohibitin (MS-261-P1, NeoMarkers), anti-GAPDH (2118, Cell Signaling), anti-CyPD (ab110324, Abcam), anti-ANT2 (14671, Cell Signaling), anti-ANT3 (PA5-35113, ThermoFischer), anti-OXPHOS (ab110411, Abcam), anti-HKII (sc130358, Santa Cruz), anti-UQCRC1 (sc65238, Santa Cruz), anti-Grim19 (sc271013, Santa Cruz), and anti-SDHA (sc166947, Santa Cruz). Statistical comparison of data was assessed with the two-sided Student’s t-test.
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